WO2011011973A1

WO2011011973A1 - Fusion protein of anti-cd20 antibody fab fragment and lidamycin, preparation method and use thereof

Info

Publication number: WO2011011973A1
Application number: PCT/CN2010/001141
Authority: WO
Inventors: 杨纯正; 甄永苏; 熊冬生; 邵荣光; 朱祯; 苗庆芳; 程昕; 张胜华; 许元富; 房虹; 高瀛岱; 金莲舫
Original assignee: 中国医学科学院医药生物技术研究所; 中国医学科学院血液学研究所
Priority date: 2009-07-28
Filing date: 2010-07-27
Publication date: 2011-02-03
Also published as: CN101967192A; CN101967192B; US20120195895A1

Abstract

Provided are a fusion protein of anti-CD20 antibody Fab fragment and lidamycin apoprotein (LDP) as well as coding gene, vector and host bacteria thereof; and the fusion protein functionally conjugated with lidamycin chromophore AE and method for the production thereof. Also provided are a pharmaceutical composition comprising said fusion protein and use of said fusion protein.

Description

Anti-CD20 antibody Fab fragment fusion protein with lidamycin,

Preparation method and use thereof

The invention relates to the field of oncology and biopharmaceutics. In particular, the present invention provides a fusion protein which can produce a tumor-targeting killing effect, a preparation method thereof and use thereof, and further provides an excellent drug candidate for targeted therapy of tumors. technical background

Non-Hodgkin's lymphoma (NHL) is a malignant tumor originating from lymphoid tissues, and its morbidity and mortality ranks fifth in malignant tumors. Although conventional radiotherapy and chemotherapy have higher efficiency against NHL, they have poor selectivity. While killing tumor cells, they may damage certain types of normal cells in the body, and often have obvious toxic side effects. Therefore, tumor targeted therapy has become An important way to improve the treatment effect.

In tumor-targeted therapy, the target selection of targeted therapy is very important. It is known that most NHL originates from B lymphocytes, and more than 95% of B-cell NHL expresses CD20 antigen, while CD20 is only in pre-B lymphocytes. Expression in mature B lymphocytes, mature B lymphocytes, and activated B lymphocytes; no expression in plasma cells, lymphatic pluripotent stem cells, and other tissues, and CD20 antigens are exposed, and there is no free CD20 in human serum, so CD20 It can be used as an effective target for the treatment of B cell lymphoma.

The most used in targeted therapy are monoclonal antibodies associated with tumor-associated antigens. Since the US FDA approved the anti-CD20 human mouse chimeric antibody rituximab (Ri tuximab) in 1997, people have great expectations for antibody drugs. In addition, anti-CD20 antibodies have recently been used to treat autoimmune diseases such as: rheumatoid Arthritis, systemic lupus erythematosus, etc. have become new research hotspots, which also suggests that the therapeutic application range of anti-CD20 antibodies will be further expanded. However, the number of cases treated with monoclonal antibodies increased Plus, the problem of drug resistance is becoming more and more obvious. The I-labeled anti-CD20 murine antibody Bexxar and the ⁹ °Y-labeled Zevalin antibody developed in different ways, which are different from rituximab antibody, overcome drug resistance, but have high immunogenicity only once. Injection, and the toxicity and side effects are large, and the patient is poorly tolerated. Therefore, the development of small, highly effective anti-targeting drugs targeting CD20 has become a top priority. Brief description of the invention

In response to this situation, the applicant envisages a strategy of combining miniaturized CD20 antibody Fab fragments (target vectors) with potent anti-tumor drugs (warheads).

In making miniaturized antibody fragment selection, Applicants selected anti-CD20 antibody Fab fragments as targeting vectors. The Fab fragment is composed of a heavy chain variable region VH, a CH1 in the constant region, a light chain variable region VL, and a constant region CL. It has a small molecular weight, strong penetrability, short half-life in vivo, and is easy to genetically engineer and pass. The advantages of mass production of bacterial fermentation. Moreover, due to the small molecular weight of Fab, low immunogenicity, it is not easy to produce HAMA reaction, and it can easily penetrate the dense tumor cell gap barrier and enter the deep part of solid tumor. Meanwhile, due to the lack of Fc fragment, Fc-mediated receptor binding is avoided. It is rapidly concentrated to the target site; in addition, it is easy to be genetically engineered in vitro, and the active protein gene is ligated to the Fab gene after gene encoding and expressed in the receptor to generate a targeted fusion protein. Therefore, antibody Fab fragments have attractive prospects as carriers for tumor targeting drugs.

In the "warhead" selection, the applicant selected the highly active "warhead" drug lindamycin (LDM), also known as C-1027 or C1027, which was isolated from the soil of Qianjiang County, Hubei Province, China. The enediyne antibiotic produced by Streptomyces globi sporus (CMSCC No. 0704) is the macromolecular peptide antitumor antibiotic which has been reported to have the strongest killing effect on tumor cells. LDM consists of two parts: one is an enedicyne structure chromophore (act ive enediyne, AE ), which has a cytotoxic effect but is unstable; the other is a prosthetic protein (LDP) consisting of 110 amino acid residues, which protects the chromophore from stability. The chromophore and the prosthetic protein are bound by non-covalent bonds, and the combination of the two is specific and robust, and the pro-base protein and chromophore of LDM can be resolved and molecularly reconstructed. LDM is suitable as a "warhead" drug with its unique molecular structure.

Applicants amplified the CHI fragment from the recombinant plasmid pCANTAB 5E Fcd20 Fab' containing the anti-CD20 antibody Fab fragment by genetic engineering method, and amplified the LDP gene from the shield pET30sngr ldp (Accession No. CGMCCNo. 2010), and then passed The Fab-LDP gene was obtained by S0E-PCR, and then the fragment was reassembled into the plasmid pCANTAB 5E Fcd20 Fab' excised with the CH1 gene to obtain the shield particle pCANTAB 5E-Fab-LDP containing Ant i-CD20 (Fab)-LDP. The plasmid was transduced into the expression host strain, and the culture condition was optimized by changing the culture temperature, the medium composition and the culture time, and the soluble expression Fab-LDP fusion protein was obtained, and the fusion protein and the AE molecule were reassembled to obtain enhanced fusion. Protein Fab - LDM. In an animal test, the enhanced fusion protein Fab-LDM of the present invention retains the targeting of anti-CD20 antibody and the killing activity of LDM, and the Fab-LDM of the present invention exhibits a higher tumor than the same dose of Fab, LDM. Inhibitory effect. Thus, a novel candidate targeted tumor therapeutic agent for tumor therapy, which is a fusion of an anti-CD20 antibody Fab fragment and lidamycin, is provided. DRAWINGS

Figure 1 shows the results of restriction endonuclease analysis of the recombinant expression plasmid pCANTAB 5E-Fab-LDP, wherein 1 is the DNA molecular weight standard; 2 is the recombinant plasmid pCANTAB 5E-Fab-LDP / apa l+sphl.

Figure 2a shows the results of SDS-PAGE analysis of the fusion protein Fab-LDP expression product, wherein

1 is the recombinant strain pCANTAB 5E- Fab- LDP periplasmic cavity protein after non-reductive purification; 2 is a recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein non-reducing effluent;

3 is the recombinant strain pCANTAB 5E- Fab-LDP periplasmic cavity protein non-reduction before loading; 4 is the recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein i3L original purification; 5 is the recombinant strain pCANTAB 5E-Fab-LDP week The luminal protein reduces the effluent; 6 is the recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein before the reduction is loaded. Figure 2b is a result of Western blot analysis of the fusion protein Fab-LDP expression product, wherein

1 is the recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein non-reductive purification; 2 is the recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein non-reducing effluent; 3 is the recombinant strain pCANTAB 5E-Fab-LDP periplasm Cavity protein is not restored before loading.

Figure 3a shows the results of FACS analysis of the binding activity of anti-CD20 Fab and Fab-LDP to Ra j i cells, wherein ▼ represents Fab-LDP; □ represents anti-CD20Fab

Figure 3b shows the results of in vivo imaging of dynamic distribution of anti-CD20 Fab and Fab-LDP tumor-bearing mice.

Figure 4a shows the cytotoxic effect of the enhanced fusion protein Fab-LDM on Ra j i cells. among them,

→- Fab-LDM

→■ LDM

+ ADR _β

Figure 4b shows the cytotoxic effect of the enhanced fusion protein Fab-LDM on Daud i cells. among them,

Temple LDM

- - Fab-LDM

ADR Figure 4c shows the cytotoxic effect of the enhanced fusion protein Fab-LDM on K562 cells. Its Medium,

-ψ- Fab-LDM

-Β- LDM

ADR Figure 4d shows the comparison of IC50 of different fusion cells between the fusion protein Fab-LDM and LDM. among them,

□ Fab-LDM

^ LD

Figure 5a shows mesh reinforcing _ADR β fusion protein therapeutic effect of Fab-LDM trees early murine transplanted CD20-positive B cell lymphoma model, wherein

* stands for PBS;

Representative of the country against CD20Fab 4nmol/kg;

A f^Fab-LDM 2nmol/kg;

♦i^LDM 4nmol/kg;

▼i^Fab-LDM 4nmol/kg;

〇 represents LDM 2nmol/kg.

Figure 5b shows the therapeutic effect of the enhanced fusion protein Fab-LDM on the late rat transplanted CD20+ B cell lymphoma model. among them,

• Represents PBS;

. Represents anti-CD20Fab 4nmol/kg;

Country Representative Fab-LDM 2nmol/kg;

♦ Represents LDM 4nmol/kg;

▲ stands for Fab- LDM 4nmol/kg;

▼ represents LDM 2nmol/kg.

Figure 6a shows the effect of the enhanced fusion protein Fab-LDM on body weight of rats. among them, -HH control

- - Fab 4nmol/kg

→- Fab-LDM 2nmol/kg

Fab-LDM 4nmol/kg

LDM 2nmol/kg

Let LDM 4nmol/kg Figure 6b show the effect of the enhanced fusion protein Fab-LDM on leukocytes in mice. among them,

^SZ Fab 4nmol/kg

Fab-LDM 2nmol/kg

Fab-LDM 4nmol/kg Figure 6c shows the effect of the enhanced fusion protein Fab-LDM on alanine aminotransferase (ALT) in mice. among them,

^ Fab 4nmol/kg

Fab-LDM 2nmol/kg

Fab-LDM 4nmol/kg Figure 6d shows the effect of the enhanced fusion protein Fab-LDM on aspartate aminotransferase (AST) in rats. among them,

E3 Fab 4nmol/kg

Fab-LDM 2nmol/kg

诵 Fab-LDM 4nmol/kg

Detailed description of the invention

The terms mentioned in the present invention are understood by the following definitions.

The "LDM" mentioned herein is equivalent to "LDP-AE", "Lidamycin", "Lidamycin prosthetic protein, in which a chromophore AE is bound to a prosthetic protein". The chromophore and the prosthetic protein are combined by non-covalent bonds, and the combination of the two is specific and robust. The prosthetic proteins and chromophores of LDM can be resolved and molecularly reassembled.

"AE" as referred to herein means a chromophore having a chemical structure represented by the following formula I,

Chemical name of LDM^ chromophore:

(2R, 7S, 9R, 10R) -7-amino-7,8-(2*-chloro-6*-hydroxy-1*,4*-phenylene)-10-(4,-deoxy-4 , -diaminoamino-5,,5,-dimethyl-pyranosyl)-4,8-oxa-5-oxo 1,11,13-triene-15,18-diyne-three Ring [7, 7, 3, 0 ¹⁰ ' ¹⁴ ] -2 -nonadecanol-2",3"-dihydro-7"-decyloxy-2"-methylene- 3"-oxo- , 4"-benzoxanth-5"-carboxylate. Molecular formula: C ₄₃ H ₄₂ 0 ₁₃ N ₃ C1

It is produced by a wild-type lidamycin-producing bacterium, naturally binds to a prostaglandin protein of lidamycin, and can be obtained in a free state by treating lidamycin with an organic solvent such as cold sterol at a low temperature. The chromophore AE, the free chromophore AE can be LDP-removed with AE-resistant Lidamycin prosthetic protein or genetically engineered LDP (which can be fused with other protein fragments or not fused with other protein fragments) Assembly in the same form as the native lindamycin form under low temperature conditions, this reassembly is referred to as "enhancement". "Fab" as referred to herein is equivalent to "Anti-CD20 (Fab), "anti-CD20 Fab", "Fab fragment of anti-CD20 antibody", which is shown in positions 24-46 of SEQ ID NO: 1. Anti-CD20 antibody Fab fragment".

The "Fab-LDP" mentioned in this paper is the same as "antiCD20(Fab)-LDP",

"Anti-CD20 (Fab)-LDP", "a fusion protein of a Fab fragment of an anti-CD20 antibody and a lindamycin prosthetic protein", which is a fusion protein of SEQ ID NO: 1.

"Fab - LDM" as referred to herein is equivalent to "enhanced fusion protein Fab - LDM""anti-CD20Fab-LDM" ^w Anti-CD20 (Fab) -LDM", "anti-CD20 antibody Fab and lidamycin" A fusion protein" which is a fusion protein of SEQ ID NO: 1, wherein a chromophore AE is bound to the lidomycinmycin prosthetic protein.

A fusion protein selected from one of the following sequences:

(a) an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 1;

(b) an amino acid sequence consisting of the amino acid sequence having the biological function of the amino acid sequence of SEQ ID NO: 1 and having 95% or more homology with the amino acid sequence of SEQ ID NO: 1;

(c) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 1 having the biological function of the amino acid sequence shown by SEQ ID NO: 1, and having one or several amino acids substituted, deleted or added.

2. The fusion protein of item 1, which also functionally binds to the chromophore AE of the structure shown by formula (I):

Lidamycin chromophore structure

(I)

3. A nucleic acid molecule encoding the gene for the fusion protein of item 1, which is selected from one of the following sequences:

(a) the nucleotide sequence of SEQ ID NO: 1 encoding SEQ ID NO: 1;

(b) a nucleotide sequence encoding the amino acid sequence of (b) in item 1;

(c) a nucleotide sequence encoding the amino acid sequence of (c) in item 1;

(d) A nucleotide sequence which differs from SEQ ID NO: 2 by the degeneracy of the codon, but encodes the same amino acid sequence as the sequence shown in SEQ ID NO: 2.

4. A vector operably linked to the nucleic acid molecule of item 3.

5. The vector of item 4, the vector being a plasmid.

6. A host strain comprising the vector of item 4.

7. The host strain of Project 6, which is deposited under the accession number CGMCC No. 3125, was sent to the China General Microorganisms Collection and Management Center on June 17, 2009, and named Escherichia coli named IHPAYZ.

8. A method of preparing a fusion protein of item 2, comprising the steps of:

(a) operably linking the Fab gene of the anti-CD20 antibody and the lidamycin prosthetic protein LDP gene to the plasmid pCANTAB 5E to obtain the recombinant expression plasmid pCANTAB 5E-Fab-LDP, (b) Inducing expression of the fusion protein Fab-LDP in E. coli HB2151,

(c) purifying the fusion protein obtained in step (b),

(d) assembling the fusion protein obtained in the step (c) with the chromophore of the formula (I),

(e) Optionally, it further comprises the step of determining the biological activity of the assembled fusion protein in step (d).

9. A pharmaceutical composition comprising a pharmaceutically effective amount of the fusion protein of item 1 or 2, optionally further comprising a pharmaceutically acceptable adjuvant.

10. Use of the fusion protein of item 1 or test 2 in the preparation of a medicament for tumor targeted therapy.

11. Use of item 10 for the targeted killing of lymphoma cells.

12. Use of item 10, where the lymphoma is a mouse lymphoma or a human B cell lymphoma.

13. A method of treating a disease comprising administering to a subject in need thereof an effective amount of a fusion protein of item 1 or 2.

14. The method of item 13, wherein the disease is CD20-positive B-cell lymph

15. The method of item 13, wherein the disease is non-Hodgkin's lymphoma or diffuse large B-cell lymphoma. The specific embodiments of the present invention are described by the following examples, which are merely intended to explain and explain the present invention, and do not limit the scope of the present invention. Equivalent variants that can be conceived by those skilled in the art in light of the known knowledge and the teachings of the prior art are included in the scope of the invention. detailed description

Example 1 Construction of recombinant expression plasmid pCANTAB 5E Fab-LDP

PCR amplification CH1:

Since the recombinant plasmid pCANTAB 5E Fcd20 Fab' contains the VH, VL and humanized CL, CH1 genes of anti-CD20 monoclonal antibody HI47, and the recombinant plasmid pCANTAB 5E Fcd20 Fab' contains only an apal restriction site in the CHI region, the applicant uses Infectious of human B-cell lymphoma by an anti-CD20 antibody and its chimeric (Fab') ₂ fragment via induction. YinxingLiu, ZhenpingZhu et.al. Cancer Letters 205 (2004) 143-153) As a template, CH1 is obtained. The PCR primers were synthesized by Shanghai Yingjun Co., Ltd., and the corresponding restriction sites were introduced.

Specifically, pCANTAB 5E Fcd20 Fab' was used as a template, P1 was used as a 5, a primer, and P2 was used as a 3, a primer, and PCR amplification was carried out under the following conditions: pre-denaturation at 94 ° C for 5 minutes, then 94. C was denatured for 1 minute, annealed at 56 ° C for 1 minute, and extended at 72 ° C for 1 minute for 25 cycles. After the last cycle, extend at 72 ° C for 10 minutes. A gene fragment A (about 324 bp) of a portion of CH1 starting with the apal cleavage site was obtained. Fragment A was subjected to 1.5% agarose gel electrophoresis and recovered by Bodatek A-type gel recovery kit.

Anti-CD20Fab' upstream primer PI:

5, -GCCTCC ACC AAGGGCCCATCGGTCTTCCCC- 3 ' ( SEQ ID No: 3 ) apal drunken site

Anti-CD20Fab' downstream primer P2:

5, -CGCGCTGCCACCGCCACCTGTGTGAGTTTTGTCACAAGA-3' (SEQ ID No: 4)

PCR amplification of LDP: The recombinant plasmid pET30sngr ldp (Accession No. CGMCC No. 2010) containing the LDP gene was used as a template, P3 was used as a 5, primer, and P4 was used as a 3' primer, and PCR amplification was carried out. Reaction conditions: Pre-denaturation at 94 °C 5 Minutes, then 94. C denatured for 1 minute, 60. C annealed for 1 minute, 72. C stretched for 1 minute for 25 cycles. After the last cycle, extend at 72 ° C for 10 minutes. The gene fragment B of LDP (about 330 bp) was obtained. Fragment B was subjected to 1.5% agarose gel electrophoresis, and purified by Bodatek A-type gel recovery kit.

LDP upstream primer P3:

5, -ACAGGTGGCGGTGGCAGCGCGCCCGCCTTCTCCGTC3' (SEQ ID No: 5) LDP downstream primer P4:

5, -GCGCGCATGCTCAGCCGAAGGTCAGAGCCAC-3' (SEQ ID No: 6) s phi cleavage site

SOE-PCR amplification of Fab-LDP:

The purified fragment A (CHI) and fragment B (LDP) products were used for amplification. The reaction conditions were: denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, and extension at 72 ° C for 2 minutes for 10 cycles. A further extension of 72 ° C for 10 minutes yielded a small amount of CH1-1 inker-LDP template. After completion of the previous reaction, P1 and P4 primers were added and amplified. The reaction conditions were: denaturation at 94 °C for 1 minute, annealing at 60 °C for 1 minute, 72. C extends for 2 minutes, a total of 30 cycles, and extends for another 10 minutes at 72 °C. The Fab-LDP gene fragment C (fragment A+B, about 669 bp) was obtained.

The reaction product C was subjected to 1% agarose gel electrophoresis, and recovered and purified by a Bodatek A-type gel recovery kit. The recovered Fab-LDP fragment and the pCANTAB 5E Fcd20 Fab' vector were digested with apal and sphl, respectively, and then the reaction product was subjected to 1% agarose gel electrophoresis, and recovered and purified by Bodatek A-type gel recovery kit. The obtained vector digested product and the digested product of the target gene were used in a ratio of 1:6 with Takara. The company's T4 ligase 16 After 16 hours of C-ligation, the competent E. coli HB2151 was transformed, and the recombinant cloning plasmid was screened, and the PCR and restriction enzyme digestion were performed, and sequencing. The results showed that the digestion results and sequencing results of the fusion gene recombinant expression plasmid were expected. Completely identical, the sequence is correct, named pCANTAB 5E Fab-LDP, the electropherogram of the bacterial PCR product is shown in Figure 1.

Wherein the protein sequence of Fab-LDP is as shown in SEQ ID NO: 1, wherein position 1-23 is a signal peptide; 24-130 is a light chain variable region; 131-236 is a light chain constant region; 237-358 The position is the heavy chain variable region; the 359-466 position is the heavy chain CH1 region; the 467-471 position is G ₄ S; and the 472-581 position is the lidamycin prosthetic protein. Wherein G ₄ S is a linker peptide consisting of 4 glycines and 1 serine.

The DNA sequence of Fab-LDP is shown in SEQ ID NO: 2, wherein: 1-69 is the peptide sequence, 70-390 is the light chain variable region gene sequence; and 391-708 is the light chain constant region gene. Sequence; 709-1074 is the heavy chain variable region gene sequence; 1075-1398 is the heavy chain CH1 region gene sequence; 1399-1413 is G ₄ S; 1414-1743 is the lidamycin prostaglandin gene sequence ; 1744-1746 is the stop codon. Example 2 Expression of Fab-LDP and Face Syndrome

2. 1 Fab-LDP expression

A single colony of Escherichia coli HB2151 containing plasmid pC ANT AB 5 E Fab-LDP obtained in Example 1 was inoculated into 5 ml of 2 χ ΥΤ medium containing ampicillin (Amp) 100 g/ml, and shaken at a constant temperature. In a box of 37 * C, 200 rpm, shake overnight; transfer 500 ml of Amp 100 μg / ml of 2 χ YT medium (1 liter of 2 χ ΥΤ medium containing 1. 6% trypsin, 1. 0% yeast pumping Extract, 0.5% sodium chloride, ΡΗ 7. 4), 37 Ό, 200 rpm, shake culture for 8 h, collect the cells at 6000 rpm, 4 centrifugation for 10 minutes on a low-temperature high-speed vacuum centrifuge, and resuspend the cells in 1000 ml of 2 χ medium containing Amp 100 μg/ml and 1 mM IPTG, 30 °, 200 rpm, shaking culture for 4 h; 8, 000 rpm, The cells were collected by centrifugation for 10 minutes and frozen in a -20 refrigerator for use.

The frozen cells were frozen and added with 50 ml of bacterial periplasmic protein extract (trihydroxyguanidinomethane 25 mmol / L, ethylenediaminetetraacetic acid 1 mmol / L, phenylmethylsulfonyl fluoride (PMSF) O. Lmmol/L, sucrose 20% (w/w, NaCl 200 mmol / L, pH 7.5), vortex and mix, placed in 4 lightly shake for 1 h. Centrifuge at 12000 rpm, 4 C for 20 minutes, take the supernatant. After dialysis for 12 h in PBS, purification was performed on a fast protein chromatograph (FPLC) using binding buffer (0.01 mol/L NaH ₂ P0 ₄ , 0.01 mol/L Na ₂ HP0 ₄ , 0.005% NaN ₃ , pH 7.0). Balance the affinity column, load, rinse the baseline with binding buffer, and after the baseline is stable, elute with elution buffer (0.1 mmol/L Glycine, pH 3,0), and neutralize the eluate. The pH was neutralized by flushing (1 mol/L Tris-HCl, 0.05% NaN ₃ , pH 8.2). The expression of the foreign protein was analyzed by 12% SDS-PAGE. The results showed that the induced recombinant strain expressed a large amount of expression. The source protein, and the expression product of Fab-LDP is mainly present in the bacterial periplasmic cavity (Fig. 2a).

2. 2 Confirmation of Fab-LDP by Western blotting

The protein obtained in 2.1 was subjected to electrophoresis, and the electrophoresed gel was subjected to semi-dry electrorotation in a Bio-Rad electrorotation tank under the following conditions: a constant current of 0.7 mA/cm ² and a time of 5 hours. The polyvinylidene fluoride membrane (PVDF) after the end of electroporation was incubated with a primary antibody diluted 10 times in blocking solution, anti-LDP monoclonal antibody (Accession No. CGMCC No. 1849), labeled with horseradish peroxidase (HRP). The goat anti-mouse IgG antibody was a secondary antibody and subjected to chromogenic analysis. The results showed that the recombinant strain expressed the recombinant fusion protein Fab-LDP with LDP at the C-terminus (Fig. 2b). The Escherichia coli containing the expression fusion protein Fab-LDP containing the plasmid pCANTAB 5E Fab-LDP was named IHPAYZ, and was sent to the General Collection of Microbial Culture Collection of China on June 17, 2009 for preservation. Number: CGMCC No.3125. Example 3 Immunological activity of Fab-LDP

The in vitro immunofluorescence binding activity of Fab-LDP was determined by flow cytometry. The ¹¹⁰⁶ th Raji cells were resuspended with different concentrations of FITC-labeled anti-CD20Fab fragment (Inhibition of human B-cell lymphoma by an anti-CD20 antibody and its chimeric (Fab ') 2 fragment via induction of apoptosis. YinxingLiu, ZhenpingZhu et.al. Cancer Letters 205 (2004) 143-153) or Fab-LDP obtained in Example 2 in 100 μL PBS, placed for 1 h, 2000 g, centrifuged for 10 minutes, discarded the supernatant, washed with PBS Three times, FACS measures the positive rate of anti-CD2 OFab fragment or Fab-LDP binding to Ra ji cells. It was demonstrated that the same concentration of the anti-CD20 Fab fragment and the binding activity of Fab-LDP to Raji cells were substantially the same, and the Fab-LDP fusion protein retained the ability to specifically bind to the target antigen.

FITC labeling of Fab-LDP: The Fab-LDP (5 mg/ml) to be cross-linked was dialyzed against the cross-linking reaction solution 4 X: 3 times to pH 9.0. Preparation method of cross-linking reaction solution: 7.56 g NaHC0 ₃ , 1.06 g Na ₂ C0 ₃ , 7.36 g NaCl, and make up to 1 L with water. FITC was dissolved in DMSO at a concentration of 1 mg/ml. The FITC used for each cross-linking should be freshly prepared and stored in the dark. According to the ratio of P: F (protein: FITC) =1 mg: 150, add FITC slowly to the Fab-LDP solution, gently shake it while adding it, and mix it with Fab-LDP to the flat shaker. In the dark, shake gently for 12 hours. Add 5 mol/L NH ₄ C1 to a final concentration of 50 mmol/L, and terminate the reaction for 4 h at 4 ° C. The conjugate was dialyzed in PBS for more than 4 times until the dialysate was clear. Identification of the cross-linker: protein concentration ( Mg/ml) -[A280 - 0.31 X A495]/1.4; F/P ratio: 3.1 χ A495/ [A280 - 0.31 A495] , the value should be between 2.5 and 6.5.

The in vivo immunological binding activity of Fab-LDP was determined by a small animal in vivo imager. Take 5-6 weeks BALB/c 73i//2i rats, female, weight 18-20g (purchased from Beijing Weitong Lihua Experimental Animal Center, animal certificate number: SCXK (Beijing) 2007-0001). After systemic irradiation with Cs-ray (dose: 4 Gy/only), Raji or K562 cells were inoculated subcutaneously in the right axilla. The number of cells per inoculation was 2 X 1070. When 2 ml of _β tumors grew to 60-80 mm ³ , Fab-LDP, anti-CD20 Fab, and LDP (16 nmol/head) were injected into the tail vein. The dynamic distribution of Fab-LDP in tumor-bearing mice was observed at 1 h, 3 h, and 10 h, respectively. It was found that Fab-LDP or anti-CD20 Fab can specifically bind to the tumor tissue at the CD20 antigen within 1 h; after 3 h, the tumor tissue center of the tumor-bearing mouse has begun to have Fab-LDP or anti-CD2 OFab enrichment, indicating The mAb has entered the center of the tumor; Fab-LDP or anti-CD20 Fab enrichment is still visible in the tumor tissue at 10 h, whereas LDP does not specifically bind to the tumor tissue site expressing the CD20 antigen (Fig. 3b). Example 4 Preparation of Lidamycin and Determination of Relative Content of Active Chromophore AE 4. Preparation of Lida Zunsu (LDM)

Adding 0.7 ml of saline-free bacteria to the cold-dried tube of Litamycin-producing bacteria (CGMCC NO. 0135) to form a bacterial suspension, inoculated with the golden ear to culture in Gao's No. 1 slant medium, 28, 7- 10 days, the surface grows white aerial hyphae, a small piece is inoculated in a first-stage seed 100 ml / 500 ml flask culture (fermentation medium composition: starch 1%, corn syrup 0. 5%, blood stasis 0. 5%, glucose 0. 5%, MgS0 ₄ 0. 02% KI 0. 06%, cornmeal 1. 5%, CaC0 ₃ 0.4%, tap water preparation, pH 7. 0, 15 lbs disinfection), 28, rotation Shake culture for 48 h, as a secondary seed in 5% in 1000 ml/5000 ml vial, cultured in the same fermentation medium, 28 Ό, cultured on a reciprocating shaker for 18 h, and on a 200 L fermentor, the loading was 100%, inoculation amount 2%, plus 0. 03% foaming agent for defoaming agent, tank pressure 0. 04, 28 °C, stirring 400 rpm, gas flow 1/1, pH 6. 5-7. 0, Fermentation for 96 h gave the desired fermentation broth. Take 10 L of fermentation broth, centrifuge to take the supernatant, adjust to pH 4. 0 with HC1, add (NH ₄ ) ₂ S0 ₄ 4. 5 Kg for 3 h, and centrifuge the separated force (4 Ό, 8000 rpm). /min, 15 min), the resulting precipitate was dissolved in 200 ml of cold water, dialyzed, and then centrifuged to remove insoluble matter. The supernatant was adsorbed on a hydroxyapatite column, and washed with 0. 001 M phosphate buffer (pH 6. 8). The active part was freeze-dried to obtain a crude product of 1500 mg. The crude product is soluble in water, via Sephadex G-75 Column chromatography, after lyophilization of the active fraction, gave 145 mg of anti-tumor high activity lidamycin white powder refined product.

4. 2 Determination of relative content of active chromophore AE

Compared with the LDM protein fraction, the chromophore has a smaller molecular weight and its theoretical content accounts for only 7.4% of lindamycin. Since AE is the active part of LDM, the prosthetic protein only protects the function of AE. Therefore, by measuring the relative content of AE in the total amount of chromophores, the activity of LDM products can be determined.

L Analysis of LDM by HPLC can measure the percentage of AE in the total amount of chromophores. The specific method is:

The LDM preparation prepared as described above was dissolved in an HPLC mobile phase (acetonitrile: water: 23:77), and separated on a FPLC fast egg white iridometer by Waters semi-radial C4 semi-preparative column. The eluent was acetonitrile: Water (23: 77), collected by an automatic collector, and the collected components were detected using an HPLC C4 analytical column.

The results of the analysis showed that the AE component of the LDM prepared by the inventors accounted for 90.63% of the total amount of LDM^L chromophore. The LDM product was analyzed to meet the LDM quality control standard, and the AE high-content LDM product was lyophilized and stored in a refrigerator to enhance the preparation of the fusion protein Fab-LDM.

4. 3 Preparation of the fusion protein Fab-LDM.

Take high-activity LDM lyophilized product 10 mg, add 5 ml cold methanol and shake for 5 minutes, -20 °C for 1 hour, shake in the middle for 1 time; at 0. C, centrifugation at 12,000 rpm for 20 minutes, the supernatant containing the chromophore AE, the sediment was a peptide chain, and the extraction was repeated twice. The methanol containing the chromophore AE was concentrated by evaporation, -70. C storage. The chromophore AE is unstable, and the experiment requires low temperature (4 ° C) and is protected from light.

Then, the Fab-LDP fusion protein obtained in Example 2 was separately dissolved in PBS, and a 5-fold molar chromophore-sterol solution (volume ratio of 50:1) was added thereto, and the mixture was shaken and allowed to stand at room temperature for 12 hours. Finally, the mixture was subjected to PD-10 column chromatography through A280 nm and A343. The enhanced fusion protein Fab-LDM was collected after nm UV monitoring. The Fab-LDMs referred to in the following examples were all prepared according to the present method. Example 5 Specific cytotoxicity of Fab-LDM on tumor cells

5. 1 Specific cytotoxicity of Fab-LDM on tumor cells cultured in vitro The cytotoxic effect was determined by the thiazolyl blue (MTT) method. Take logarithmic growth phase of Ra ji or Daudi cells, count, 2 χ 10 ⁴ / wells in 96-well plates, at 37. After incubating for 12 hours in a C-containing C0 ₂ incubator, different concentrations of the drug Fab-LDM were added, and each drug concentration was set to 3 parallel wells. The culture was continued for 48 hours, centrifuged at 2000 rpm/min for 10 minutes, and the supernatant was discarded. Add 20 μl of PBS dissolved MTT (5 mg/ml) to each well, continue to incubate at 37 °C for 4 hours, centrifuge at 2000 rpm/min for 10 minutes, gently discard the supernatant, and add 100 μl of dimethyl sulfoxide (DMS0). ), shaking at room temperature for 5 minutes on a shaker, and measuring the absorbance at 546 nm on a microplate reader. In each experiment, there were 3 wells of drug-free control wells and cell-free control wells. Calculate cell viability and half inhibition concentration (IC ₅ ) by the following formula:

Cell viability = (A dosing group - A blank group) / (A control group - A blank group) fusion protein was found to strengthen the Fab-LDM on Raj i, Daudi cells IC _5. Values are 0. 9x10- ^1Q M and 0. 8x10-, IC ₅ K562 cells no specific CD20 antigen. Is 3. 0 χ10 ^_10 Μ, Lidamycin IC ₅ and the pair of Raj i, Daudi cells. The values are 3. 1x10 - ^1D M and 2. 9x10 - , IC ₅ for K562 cells without specific CD20 antigen. The value is 2. 8 χ ¹⁰ " ¹⁰ Μ, indicating that Ant i-CD20 (Fab) -LDM retains the cytotoxicity of lidamycin and has selective killing effect on tumor cells. The specific results can be seen in Figure 4a. -d.

5. 2 Specific cytotoxicity of Fab-LDM on primary tumor cells of patients The cytotoxicity was determined by the MTT assay. Take the patient's primary tumor cells, count, 2X10 ⁴ /well in 96-well plates, at 37. C culture in an incubator containing 5% C0 ₂ After the hour, different concentrations of the drug Fab-LDM were added, and each drug concentration was set to 3 parallel wells. The culture was continued for 72 hours, centrifuged at 2000 rpm/min for 10 min, and the supernatant was discarded. Add 20 l of PBS dissolved MTT (5 mg/ml) per well, 37. C continued to culture for 4 hours, centrifuged at 2000 rpm / min for 10 minutes, gently discard the supernatant, add 100 μl of dimercaptosulfoxide (DMS0), shake at room temperature for 5 minutes on a shaker, and measure 546 nm light on a microplate reader. Absorbance. In each experiment, there were 3 wells of drug-free control wells and cell-free control wells. According to the formula: cell viability = (A plus group - A blank group) / (A control group - A blank group) χ 100%, calculate cell viability and half inhibition concentration (IC ₅ ) value.

It was found that Fab-LDM retained the cytotoxicity of lidamycin and selectively inhibited the primary tumor cells of patients. It is worth mentioning that the primary cells of patients with resistance to rituximab are also very Good selective killing effect (Table 1).

Table 1: Growth inhibition of primary tumor cells in NHL patients by fusion protein Fab-LDM in vitro

Abbreviations Note: NHL stands for non-Linkin's lymphoma; DLBCL stands for diffuse large B-cell lymphoma. Example 6 Treatment of a rat transplanted CD20+ B cell lymphoma model by Fab-LDM

6. 1 Fab-LDM treatment of early transplanted CD20+ B cell lymphoma f-type

Five-week-old BALB/c mice with a well-growth of 16-18 g were randomly divided into groups. After Cs irradiation, the Raj cells in the armpits of the rats were inoculated 2 χ ^{7 7} /s within 24 h. After 7 days of feeding, the tail vein was administered intravenously, and 9 days later, the drug was administered again. The therapeutic effect of Fab-LDM on the subcutaneous lymphoma of the mouse was observed, and the growth curve of the mouse tumor was drawn. 2 ml每应。 The control group was given intravenous saline 0.2 ml / only. The other groups were given different doses of the fusion protein Fab-LDM, LDM and 4 nmol/kg of anti-CD20 Fab, all of which were injected into the tail vein, 0.2 ml/mouse. During the test, the long diameter a and short diameter b of the tumor were measured every three days, and the animal body weight was recorded. The tumor volume was calculated by the formula V-0. 5ab ² and the tumor inhibition rate was calculated (Fig. 5a).

The results of the enhanced fusion protein Fab-LDM showed that Fab-LDM in both 2nmol/kg and 4nmol/kg groups significantly inhibited or delayed the growth of transplanted lymphoma in mice, and showed a free force compared with the corresponding dose. The stronger tumor growth inhibition effect of mycin increases the therapeutic effect of lindamycin. On the 39th day of the experiment, the tumor inhibition rates of Fab-LDM in the 4nmol/kg and 2nmol/kg dose groups were 88% and 73%, respectively, which were stronger than the corresponding doses of the LDM group, 49% and 69%. rate. During the experimental treatment, the animals gained weight and were generally in good condition, indicating that the animals were tolerant to the given dose (Table 2). Table 2. Growth inhibition of Fab-LDM on transplanted CD20+ B cell lymphoma in early rat

, compared with LDM?f, AO. 05, . Compared with the blank control *(0. 05, . . compared with the blank control AO. 01 ,

6. 2 Fab-LDM treatment of advanced rat transplanted CD20+ B cell lymphoma model. A 5-week BALB/c mouse with a good growth condition of 16-18 g was randomly grouped. After Cs irradiation, the Raj cells in the armpits of the rats were inoculated 2 χ 10 ⁷ /s within 24 hours. After 25 days, the tail vein was administered intravenously, and 9 days later, the drug was administered again to observe the subcutaneous lymphoma of Fab-LDM. Therapeutic effect, plotting the growth curve of mouse tumors (Fig. 5b). 2 ml每应。 The control group was given intravenous saline 0.2 ml / only. The other groups were given different doses of the enhanced fusion protein Fab-LDM and LDM, both of which were injected into the tail vein, 0.2 ml / each. During the test, the long diameter a and the short diameter b of the tumor were measured every three days, and the animal body weight was recorded. The tumor volume was calculated by the formula V = 0.55 ² and the tumor inhibition rate was calculated. The results of the enhanced fusion protein Fab-LDM showed that Fab-LDM in 2mnol/kg and 4nmol/kg groups significantly inhibited or delayed the growth of transplanted lymphoma in mice, and showed a dose-free force. Dacmycin has a stronger tumor growth inhibition effect, showing a good therapeutic effect of Fab-LDM on advanced lymphoma. The results of the experiment on the 47th day showed that the tumor inhibition rates of Fab-LDM in the two dose groups of 4nmol/kg and 2nmol/kg were 79.2% and 67.5%, respectively, which was stronger than the corresponding dose of LDM group 52.1%. And 65% inhibition rate (Table 3). During the experimental treatment, the animals gained weight and were generally in good condition, indicating that the animals were tolerant to the given dose.

Inch

Table 3. Growth inhibition of Fab-LDM on advanced rat transplanted CD20+ B cell lymphoma

Group punishment amount of mice weight (g) tumor volume inhibition rate

(nmol/kg) (mm ³ ) X (%)

Start / end start / end .

Control 5/5 16. 5/20. 4 3255. 76

2 5/5 16. 8/20. 6 1559. 52 52. 1 mouth

LDM

4 5/5 16. 5/20. 4 1136. 86 65 ^πα

2 5/5 16. 2/20. 5 67. 5 ^D *

Fab-LDM

4 5/5 17. 3/20. 6 79. 2°*

* With LD Curry, KQ. 05, . Compared with the blank control, AO. 05, . . Compared with the blank control AO. 01. Example 7 Toxicity Assessment of Fab-LDM

7. 1 weight and survival

The changes in body weight and survival were observed, and adverse reactions or toxicity with significant physiological significance such as sputum dander, dehydration, lethargy, ataxia, and shortness of breath were recorded. The body weight of the mice was weighed each time to assess the toxicity of the drug. As a result, no adverse reactions with significant physiological significance such as sputum dander, dehydration, lethargy, ataxia, and shortness of breath were observed in the entire treatment window. After treatment, the PBS, Fab-LDM 2nmol/kg, Fab-LDM 4 leg 01/1^ and 0)1 1111101 /1^ groups of the mouse-resistant xenograft model showed no decrease in body weight and gradually increased. The LDM 4nmol/kg group showed a decrease in body weight (Fig. 6a).

7. 2 Pathological statistics of the injury site

At the end of the experiment, the rats were sacrificed and organs such as heart, liver, spleen, lung, kidney, small intestine were taken, and formalin was soaked overnight and stained with HE. Using the LEICA QWINV3 system, 10 slices of 50 fields were statistically analyzed for multiple organs in each treatment group, and the damage area of each organ in each treatment group was calculated. PBS, Fab-LDM 2nmol/kg, Fab-LDM 4nmol/kg and LDM2 nmol/kg showed no obvious case damage, while LDM 4nmol/kg group caused mild tubular swelling and hepatic tissue cell infiltration. Damage to spleen hematopoietic cells (Table 4). Table 4: Pathological statistics of the injury site

*: spleen injury statistics = 4 nmol / kg LDM treatment group nucleated cells / PBS treatment group nucleated cells. **: Liver (kidney) lesion area statistics = liver (kidney) lesion area / liver (kidney) total area. 7. 3 Detection of biochemical indicators such as liver function, kidney function and blood routine

At the end of the experiment, the rats were sacrificed, blood was taken from the decapitation, and blood changes were detected after partial anticoagulation treatment. Another part of the whole blood was coagulated and serum was taken to detect changes in liver function and renal function. The results showed that Fab-LDM 2nmol/kg and Fab-LDM 4nmol/kg had no effect on liver function and kidney function, and Fab-LDM 4nmol/kg group caused a slight decrease in white blood cells (Fig. 6b-d). Invention effect:

The advantages and positive effects of the present invention are: using a combination of genetic recombination and molecular reconstitution, an enhanced fusion protein Fab-LDM of anti-CD20 Fab and anti-tumor antibiotic lindamycin is prepared, which not only retains the monoclonal antibody against CD20 antigen. It also has strong tumor cell-specific killing activity, and it also shows good anti-tumor effect in vivo. It has reached a new level in the miniaturization of tumor-targeted immunotherapy drugs and has a good application prospect.

Claims

Rights request

A fusion protein selected from one of the following sequences:

(b) an amino acid sequence consisting of an amino acid sequence having a biological function of amino acid sequence of 3 £ (310^:1) and having 95% or more homology with the amino acid sequence of SEQ ID NO: 1;

2. The fusion protein of claim 1 which also functionally binds to a chromophore AE of the structure of formula (I):

3. A nucleic acid molecule encoding the gene of the fusion protein of claim 1 selected from one of the following sequences:

(a) the nucleotide sequence of SEQ ID NO: 1 encoding SEQ ID NO: 1; (b) a nucleotide sequence encoding the amino acid sequence of (b) of claim 1;

(c) a nucleotide sequence encoding the amino acid sequence of (c) of claim 1;

(d) A nucleotide sequence which differs from SEQ ID NO: 2 but which encodes the same amino acid sequence as SEQ ID NO: 2 because of codon degeneracy.

4. A vector operably linked to the nucleic acid molecule of claim 3.

5. The carrier of claim 4, said carrier being a shield particle.

6. A host strain comprising the vector of claim 4.

7. The host strain of claim 6 which is deposited under the accession number CGMCC No. 3125 and sent to the General Collection of Microorganisms Collection and Management of China on June 17, 2009, and named Escherichia coli named IHPAYZ.

8. A method of preparing the fusion protein of claim 2, comprising the steps of:

(a) The Fab gene of the anti-CD20 antibody and the lidamycin prosthetic protein LDP gene are operably linked into the plasmid pCANTAB 5E to obtain the recombinant expression plasmid pCANTAB 5E-Fab-LDP,

(b) Inducing expression of the fusion protein Fab-LDP in E. coli HB2151,

(c) purifying the fusion protein obtained in step (b),

9. A pharmaceutical composition comprising a pharmaceutically effective amount of the fusion protein of claim 1 or 2, optionally further comprising a pharmaceutically acceptable adjuvant.

10. Use of the fusion protein of claim 1 or 2 in the manufacture of a medicament for tumor targeted therapy.

11. The use of claim 10 for the targeted killing of lymphoma cells. The use of claim 10, wherein the lymphoma is a mouse lymphoma or a human B

13. A method of treating a disease comprising administering to a subject in need thereof an effective amount of the fusion protein of claim 1 or 2.

14. The method of claim 13, wherein the disease is a CD20 positive B cell lymphoma.

15. The method of claim 13, wherein the disease is non-Hodgkin's lymphoma or diffuse large B-cell lymphoma.